SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.     

High glucose increases glomerular filtration barrier permeability by activating protein kinase G type Iα subunits in a Nox4-dependent manner

Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type Iα (PKGIα). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGIα activation, and that PKGIα was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30 mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD(P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGIα into cultured podocytes. Furthermore, HG increased PKGIα dimerization by 138% (0.23±0.04 vs. 0.54±0.09; P<0.05); this effect was abolished with a siRNA against Nox4. Our observations suggested that HG could increase albumin permeability across the podocyte filtration barrier via Nox4-dependent PKGIα dimerization.     

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.     

Polysaccharides and mucin 5AC (MUC5AC) expression in gallbladder mucosa of young patients with gallstones as evaluated by spatial visualization and quantification

The study aimed at examination of tissue expression of polysaccharides and secretory mucin 5AC (MUC5AC) in young patients (up to 25 years of age) with a symptomatic gallstones. For comparison, patients most frequently subjected to cholecystectomy were studied, i.e. patients of approximately 50 years of age with the same diagnosis. In quantitative studies on tissue expression of both mucus components, the modern technique of spatial visualization was applied for the first time. Application of the technique permitted to demonstrate significant positive relationships between expression of glycoproteins (immunocytochemical ABC technique for detection of MUC5AC) and expression of sugar components in mucus (PAS technique) and to confirm suitability of the technique for quantitative appraisal of both histochemical and immunocytochemical reactions. An even higher expression of polysaccharides in the entire mucosa and of MUC5AC was detected in gallbladder epithelium of 50-year-old patients, as compared to young patients with symptomatic gallstones. In the young patients, expression of polysaccharides correlated with inflammatory activity (grading), width of gallbladder wall and PLT level in peripheral blood. A significantly higher expression of polysaccharides in gallbladder epithelium was demonstrated in young patients admitted in the emergency mode to the hospital. These correlations in young patients may suggest a role of both mucus components in pathogenesis of cholelithiasis in this age group. A quantitative appraisal of mucus component expression in the two parts of gallbladder mucosa (epithelium vs. entire mucosa) using spatial visualization technique permitted to more accurately compare production of glycoproteins and of polysaccharides in patients with cholelithiasis and to demonstrate additional correlations of a potential clinical significance.     

Cysteine peptidases and their inhibitors in breast and genital cancer

Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.     

Assessment of S100 protein expression in the epididymis of juvenile and adult European bison

In our study, we decided to compare S100 protein expression in the material obtained from the epididymes of 5- and 12-month-old calves, and adult European bison, and to detect any differences in S100 expression according to the animal age and size of the organ examined. We used the epididymes obtained from 6 adult European bison aged 6-12 years, from 6 at the age of 12 months and 6 calves aged 5 months. Immunocytochemical reactions were performed using the avidin-biotinylated-peroxidase (ABC) technique according to HSU. Specific polyclonal rabbit antiserum against bovine S100 protein (Bio Genex Laboratories) at a dilution at 1:400 was applied. We found the expression of S100 protein in endothelial cells of arteries, veins and lymphatic vessels in all the study animals. At the same time, we found no differences in the expression of S100 protein in vascular endothelial cells. Our observations seem to indicate that S100 expression in endothelial cells of European bison epididymis is not correlated with age or maturity of the organ tested. We found S100 protein in smooth muscle cells of arteries and veins in all European bison specimens examined. Interestingly in the current study, in young 5-month-old sexually immature European bison specimens we observed weaker expression of S100 protein in smooth muscle cells of small vessels as compared to the same cell type both in large vessels in these animals and in small vessels in adult specimens.     

The conformational properties of dehydrobutyrine and dehydrovaline: theoretical and solid‐state conformational studies

Dehydrobutyrine is the most naturally occurring dehydroamino acid. It is also the simplest dehydroamino acid having the geometrical isomers E/Z. To investigate its conformational properties, a theoretical analysis was performed on N‐acetyl‐α,β‐dehydrobutyrine N′‐methylamides, Ac‐(E)‐ΔAbu‐NHMe and Ac‐(Z)‐ΔAbu‐NHMe, as well as the dehydrovaline derivative Ac‐ΔVal‐NHMe. The ?, ψ potential energy surfaces and the localised conformers were calculated at the B3LYP/6‐311 + + G(d,p) level of theory both in vacuo and with inclusion of the solvent (chloroform, water) effect (SCRF method). The X‐ray crystal structures of Ac‐(Z)‐ΔAbu‐NHMe and Ac‐ΔVal‐NHMe were determined at 85 and 100 K, respectively. The solid‐state conformational preferences for the studied residues have been analysed and compared with the other related structures. Despite the limitations imposed by the C^α = C^β double bond on the topography of the side chains, the main chains of the studied dehydroamino acids are more flexible than in standard alanine. The studied dehydroamino acids differ in their conformational preferences, which depend on the polarity of the environment. This might be a reason why the nature quite precisely differentiates between ΔVal and each of the ΔAbu isomers, and why, particularly so with the latter, they are used as a conformational tool to influence the biological action of usually small, cyclic dehydropeptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Contrasting Species—Environment Relationships in Communities of Testate Amoebae, Bryophytes and Vascular Plants Along the Fen–Bog Gradient

We studied the vegetation, testate amoebae and abiotic variables (depth of the water table, pH, electrical conductivity, Ca and Mg concentrations of water extracted from mosses) along the bog to extremely rich fen gradient in sub-alpine peatlands of the Upper Engadine (Swiss Alps). Testate amoeba diversity was correlated to that of mosses but not of vascular plants. Diversity peaked in rich fen for testate amoebae and in extremely rich fen for mosses, while for testate amoebae and mosses it was lowest in bog but for vascular plants in extremely rich fen. Multiple factor and redundancy analyses (RDA) revealed a stronger correlation of testate amoebae than of vegetation to water table and hydrochemical variables and relatively strong correlation between testate amoeba and moss community data. In RDA, hydrochemical variables explained a higher proportion of the testate amoeba and moss data than water table depth. Abiotic variables explained a higher percentage of the species data for testate amoebae (30.3% or 19.5% for binary data) than for mosses (13.4%) and vascular plants (10%). These results show that (1) vascular plant, moss and testate amoeba communities respond differently to ecological gradients in peatlands and (2) testate amoebae are more strongly related than vascular plants to the abiotic factors at the mire surface. These differences are related to vertical trophic gradients and associated niche differentiation.     

Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.